The present invention is concerned with a vaccine against Lyme disease, with a process for obtaining said vaccine, with new monoclonal antibodies, with new antigens and with new recombinant DNA""s and vectors.
Lyme borreliosis is the most common infectious disease transmitted by ticks in the temperate regions. It is caused by the spirochete Borrelia burgdorferi which is transmitted to humans in particular by ticks of the genus Ixodes. The disease is a chronic, progressive infection which attacks many organs, such as the skin, the central and peripheral nervous system, the heart, the liver, the kidneys and musculoskeletal system. Since a reliable treatment of this disease by therapy with antibiotics is difficult, at the moment great efforts are being made to investigate the pathogen itself and the immune response of the host to infection with Borrelia burgdorferi. In the case of persons afflicted by Lyme disease, there is admittedly ascertained a high titre of antibodies against Borrelia burgdorferi which, however, do not provide any protection against the infection. It is assumed that the pathogen passes over very quickly from the blood circulation into the tissues and can there no longer be directly reached by the immune system. This would mean that a protection by antibodies is only possible immediately after commencement of the infection, i.e. as long as the pathogen is still present in the blood circulation.
The fact that a natural infection with Borrelia burgdorferi has been found in various kinds of animals has led to attempts to establish laboratory models for Lyme disease. This also took place with limited success. Thus, in the case of experiments which had the object of inducing in mice a specific immune response for Borrelia burgdorferi, it was found that the infection of inbred mouse strains with a prolonged cultured isolate of Borrelia burgdorferi led to moderate but significant pathomorphological changes in various organs, such as the brain, the heart, the lungs and the kidneys, which were comparable to those which are to be observed in patients with Lyme disease (see Schaible et al., Infect. Immun., 1, 41/1988). The development of a serious aspect of the disease in animals was presumably prevented either by the immune defence of the host and/or by the reduced virulence of spirochetes cultured in vitro for a comparatively long period of time (see Johnson et al., J. Clin. Microbiol., 20, 747/1984; Schwan et al., Infect. and Immun., 56, 1837/1988).
It is an object of the present invention to provide an effective vaccine against Lyme disease. However, for this purpose, it is first necessary to develop an appropriate animal laboratory model. It is now suggested that a mouse strain without functionable T- and B-cells, the so-called scid mouse (see Bosma et al., Nature, 10, 52/1983) can serve as experimental animal since scid mice, in the case of infection with a pathogenic Borrelia burgdorferi isolate, develop a multi-systemic disease, namely, mainly polyarthritis and carditis. By means of this animal model, it is possible for the first time to test the action of vaccines against Lyme disease.
One subject of the present invention is a passive vaccine against Lyme disease which contains one or more specific monoclonal antibodies for the 31 kD antigen (OspA) and/or the 34 kD antigen (OspB) of Borrelia burgdorferi and especially OspA and/or OspB of Borrelia burgdorferi of the strain B31 (ATCC 35210) and/or ZS7 (DSM 5527). A vaccine is preferred which contains one of the antibodies of the class IgG according to the present invention and especially preferably of the subclass IgG2b or IgGl. Surprisingly, in contradistinction to the administration of another antibody, for example against the 41 kD surface antigen of Borrelia burgdorferi (flagellin), the administration of the antibody according to the present invention has the result, in the case of immune-deficient experimental animals and preferably of scid mice which have been infected with viable pathogenic Borrelia burgdorferi and preferably with Borrelia burgdorferi ZS7, that the development of arthritis, carditis and hepatitis is completely or at least substantially prevented.
The vaccine according to the present invention with the antibody as active material can possibly also contain conventional carrier, filling and adjuvant materials.
Furthermore, the present invention provides a process for obtaining a passive vaccine against Lyme disease from lymphocytes or spleen cells of an experimental animal, preferably of a mouse, which has bee immunised with Borrelia burgdorferi organisms or parts thereof, preferably with complete Borrelia burgdorferi B31 and/or ZS7 organisms, in which, from the lymphocytes or spleen cells of the immunised animals, there is obtained, by cell fusion, a hybridoma which produces a monoclonal antibody according to the present invention.
Thus, a subject of the present invention is also a hybridoma cell line (ECACC 89091302) which produces an antibody LA-2 against OspA (IgG2b) according to the present invention. Furthermore, the subject of the present invention is also the hybridoma cell line ECACC 90050406 producing the antibody LA-26.1 against OspA (IgGl), as well as the hybridoma cell lines ECACC 90050405 and ECACC 90050407 producing antibodies LA-25.1 and LA-27.1, respectively, against OspB (IgG2b and IgGl, respectively).
Furthermore, the present invention provides the pathogenic Borrelia burgdorferi strain ZS7 (DSM 5527).
In addition, the subject of the present invention is an antigen which immune-reacts with a monoclonal antibody according to the present invention. By this is to be understood an antigen which contains the whole amino acid sequence of OspA or OspB or also only an immunogenically-acting part sequence (immunogenic epitope) of OspA or OspB, respectively. Potentially immunogenic epitopes of these proteins can be determined without difficulty by a structural analysis of the OspA protein, for example a Chou-Fasman analysis, and then tested experimentally for their effectiveness.
Yet another subject of the present invention is also, in particular, a recombinant antigen which immune-reacts with the antibody according to the present invention in which the DNA sequence coding for the antigen is present on a recombinant vector, preferably a prokaryotic vector, which is suitable for the protein expression.
In particular, a subject of the present invention is an antigen from Borrelia burgdorferi ZS7 which specifically immune-reacts with the antibody according to the present invention and which contains the amino acid sequence shown in FIG. 1 of the accompanying drawings or an immunogenic epitope of this sequence. Consequently, the present invention also concerns a recombinant DNA which contains (1) the sequence shown in FIG. 1, (2) a nucleic acid sequence corresponding to it in the scope of the degeneration of the genetic code or (3) one hybridising under stringent conditions with a sequence from (1) and/or (2), which sequence codes for the 31 kD antigen of Borrelia burgdorferi strain ZS7 or an immunogenic epitope thereof. The term stringent hybridising conditions is thereby to be understood as in Maniatis et al., Molecular Cloning, A Laboratory Manual (lo82), Cold Spring Harbor Laboratory, New York.
Especially preferred is an antigen according to the present invention which is a recombinant non-fusion protein or xcex2-galactosidase fusion protein.
Furthermore, the present invention is concerned with a recombinant vector which contains one or more copies of a recombinant DNA according to the present invention. The vector according to the present invention can be a prokaryotic and/or eukaryotic vector but is preferably a prokaryotic vector. The recombinant vector can be present extrachromosomally in the host cell (for example plasmid) or it can also be integrated in the genome of the host cell (for example bacteriophage lambda). The vector according to the present invention is preferably a plasmid, the recombinant vector pZS-7/31-2 (DSM 5528) being especially preferred.
The present invention also provides a process for obtaining antigens according to the present invention by investigation of a Borrelia burgdorferi gene bank with one or more antibodies according to the present invention in which the clones are isolated which show a positive immune reaction with the antibodies used.
Since the antigen according to the present invention itself also can be used for active immunisation, i.e. for the induction of antibody formation in the organism, the present invention also provides an active vaccine against Lyme disease which, as active material, contains an antigen according to the present invention, optionally together with conventional carrier, filling and adjuvant materials. A preferred embodiment is when the antigen according to the present invention is obtained gene-technologically.
Indeed, it could be shown that the administration of native or recombinant OspA to normal mice induces the formation of protective antibodies which, after passive transfer into scid mice, protect these against Lyme borreliosis. In particular, it is found that recombinant OspA induces a protective immune response comparable with native OspA and, therefore, represents a highly promising condidate for a vaccine against Lyme borreliosis in humans.
The present invention also provides a process for obtaining a passive vaccine against Lyme disease in which experimental animals, preferably mice, are immunised with an antigen according to the present invention and protective, polyclonal or monoclonal antibodies are obtained in the usual way from the immunised experimental animals.
Finally, the present invention also provides a process for the isolation and reculturing of pathogenic Borrelia burgdorferi organisms, wherein, from immune-deficient experimental animals, preferably mice, which have previously been infected with the pathogen, there is obtained the pathogen, whereby the pathogeneity of the pathogen is retained. Especially preferred is a process in which pathogenic Borrelia burgdorferi strain ZS7 (DSM 5527) organisms are obtained from the blood and/or joints of infected scid mice.